The overall goal of our laboratory is to understand the genetic and metabolic defects that produce hypercholesterolemia and atherosclerosis in man. We have defined a new pathway for the control of cholesterol metabolism in human cells. This pathway, called the low-density lipoprotein (LDL) pathway, consists of an ordered sequence of events by which cells bind LDL at a high affinity surface receptor site, internalize the lipoprotein by endocytosis, and release the lipoprotein cholesterol for use in cellular membrane synthesis. The importance of the LDL pathway is illustrated by the demonstration that mutations at two different steps in this sequence produce three important clinical disorders involving hypercholesterolemia and/or atherosclerosis in man. Since submission of the parent grant (HL-20948), we have made a major breakthrough that allows us to probe the genetics of the LDL receptor pathway in a powerful way. We have divised a method to extract the cholesteryl esters from the core of LDL and to reconstitute this core with a variety of oxogenous lipids. The reconstituted LDL retains the ability to bind to the LDL receptor of cultured cells, thereby delivering its contained lipid only to cells that possess LDL receptors. We now propose to synthesize toxic molecules that can be incorporated into LDL. Use of such reconstituted toxic LDL will permit the isolation of mutant clones of cultured cells that have blocks at specific sites in the LDL receptor pathway. A biochemical comparison of the molecular properties of such mutant clones with those of normal cells should allow the identification of hitherto unknown molecules that participate in the receptor-mediated uptake of LDL. The availability of cells with experimentally-induced mutations with blocks in the LDL pathway will make feasible a type of definitive biochemical analysis that would otherwise not be possible.